In Vitro and In Vivo Investigations into the Biocompatibility ofDiamond-Like Carbon (DLC) Coatings for Orthopedic Applications

Matthew Allen,1 Ben Myer,2 Neil Rushton2

1 Department of Orthopedic Surgery, Institute for Human Performance, SUNY–Upstate Medical University,Syracuse, New York

2 University of Cambridge Orthopaedic Research Unit, Addenbrooke’s Hospital, Hills Road, Cambridge, UK

Received 10 July 2000; revised 12 January 2001; accepted 18 January 2001Published online 00 Month 2001

Abstract: Diamond-like carbon (DLC) shows great promise as a durable, wear- and corro-sion-resistant coating for biomedical implants. The effects of DLC coatings on the musculo-skeletal system havenot been investigated in detail. In thisstudy, DLC coatingsweredepositedon polystyrene 24-well tissue cultur e plates by fast-atom bombardment from a hexaneprecursor. Two osteoblast-likecell lineswerecultured on uncoated and DLC-coated plates forperiodsof up to 72 h. Theeffectsof DLC coatingson cellular metabolism were investigated bymeasuring the production of three osteoblast-specific marker proteins: alkaline phosphatase,osteocalcin, and type I collagen. There was no evidence that the presence of the DLC coatinghad any adverse effect on any of the parameters measured in this study. In a second series ofexperiments, DLC-coated cobalt-chromium cylinders were implanted in intramuscular loca-tions in rats and in transcortical sites in sheep. Histologic analysis of specimens retrieved 90days after surgery showed that the DLC-coated specimens were well tolerated in both sites.These data indicate that DLC coatings are biocompatible in vitro and in vivo, and furtherinvestigations into their long-term biological and tribological performancearenow warranted.© 2001 John Wiley & Sons, Inc. J Biomed Mater Res (Appl Biomater) 58: 319–328, 2001

Keywords: osteoblast; diamond-like carbon; coating; biocompatibility

INTRODUCTION

Concerns over the long-term effects of total joint replace-mentscenter on the issueof particulatewear debrisgeneratedfrom the articulating or fixation surfaces of the implant.1,2 Invitro and in vivo studies have shown that particulate debrisstimulates the production of proinflammatory mediators bycells in periprosthetic tissues.1,3–5These mediators are impli-cated in the development of osteoclastic bone resorption atthe bone–cement or bone–implant interface, and hence in thepathogenesis of aseptic implant loosening.6–8

Improved biological strategies for controlling the effectsof particulate debris on periprosthetic tissues are being ac-tively researched, but improvements in the wear properties ofthematerialsused in total joint replacement havethepotentialfor far greater impact on the problem of particulate weardebris. In recent years, attention has focused on two ap-proaches to improving the performance of implant materials:

replacement (e.g., ceramic instead of metal femoralheads)9–11 and refinement (e.g., highly cross-linked polyeth-yleneacetabular liners12,13). Our laboratory hasbeen workingon methods of improving the performance of contemporarymetal components in total joint replacement, with aparticularfocus on improving the wear characteristics of femoral com-ponents from total hip and knee prostheses. This work hasutilized a surface engineering approach14 to the problem ofimplant wear, and has centered on investigations into the useof carbon-based thin filmsaswear-retardant coatings for jointprostheses. These films are produced from solid carbon orliquid/gaseous hydrocarbon sources, using either ion beam orplasma discharge deposition systems.15 The molecular ar-rangement of carbon atoms within these films can be highlyvariable, and depends on the exact deposition conditions thatare employed, but all contain a mixture of sp3 and sp2

coordinated carbon together with significant amounts of hy-drogen.16 Thephysical propertiesof thenoncrystalline, amor-phous coatings formed by ion-beam deposition lie betweenthose of graphite and diamond, leading to the introduction ofthe term “diamond-like carbon” (DLC).

The specific properties of an individual DLC coating arecritically dependent on the conditions under which it is de-posited, but typically include extreme hardness, excellent

Correspondence to: Matthew J. Allen, Vet. M.B., Ph.D., Department of OrthopedicSurgery, Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210(e-mail: allenm@mail.upstate.edu)

Contract grant sponsor: Howmedica International, Staines, UK

© 2001 John Wiley & Sons, Inc.

319

thermal conductivity, resistance to chemical attack, and goodadhesion to a variety of metallic and nonmetallic sub-strates.17,18 In addition, DLC coatings possess low coeffi-cients of friction and are highly resistant to mechanicalwear.19 DLC coatings are now widely used as scratch-resis-tant coatings for civilian and military optics,20 and as wear-resistant coatings for machine tools19 and surgical instru-ments.21

Interest in the use of DLC as a coating for implant mate-rials has developed more slowly, primarily because of con-cerns over the possibility that thin film coatings might not beable to withstand thein vivo environment, leading to theproduction of soluble or particulate wear debris. However,advances in substrate coating technology have led to dramaticimprovements in the mechanical properties of the substrate–coating interface, and modern-generation DLC coatings ad-here strongly to many of the metallic and polymeric bioma-terials currently in clinical use,20,22,23forming an inert, chem-ically resistant protective layer that should improve thetribological properties of articulating surfaces.

Although a number of laboratory-based studies haveshown that DLC coatings have the potential to improve thewear performance of current implant materials,14,24,25 lessattention has been paid to the preclinical evaluation of thebiocompatibility of DLC within musculoskeletal tissues. Thespecific aims of this study were to use bothin vitro and invivo models to investigate the effects of DLC coatings on themusculoskeletal system.

MATERIALS AND METHODS

Cell Lines

Two human osteoblast-like cell lines (MG-63 and SaOS-2)were used in this study. Both cell lines were purchased fromthe American Type Cell Culture (ATCC) through the Euro-pean Collection of Animal Cell Cultures, Porton Down.SaOS-2 cells were grown in McCoy’s 5A medium, andMG-63 cells were maintained in Dulbecco’s Modification ofEagle’s Medium. In each case, the basal medium was sup-plemented with 10% (v/v) fetal calf serum, 50 IU/mL peni-cillin, 50 mg/mL streptomycin and 2mML-glutamine. Cul-tures were placed in a 37 °C incubator with a humidifiedatmosphere containing 5% CO2 in air. Flasks were fed every3 days and split at 75% confluence using trypsin-EDTAsolution.

Experimental Animals

Sprague–Dawley rats (200–250 g body weight) and skele-tally mature ewes of mixed breeds (70–95 kg body weight)were used in this study. Animals were housed under standardconditions, and all surgical procedures were performed inaccordance with prevailing legislation in the UK.

Test Samples

Uncoated and DLC-coated polystyrene 24-well plates wereused in thein vitro experiments. The DLC coating, which hada mean thickness of 0.5mm, was deposited by fast atombombardment from an acetylene precursor (Table I).

For the surgical study, 43 10 mm cylinders of cobalt-chromium (CoCr) alloy were obtained from HowmedicaInternational (Staines, UK). Uncoated and DLC-coated spec-imens were tested. The DLC coating was deposited by radio-frequency plasma-assisted chemical vapor deposition (rf-PACVD), using an amorphous silicon interlayer to enhancecoating-substrate adhesion.20 The deposition conditions forthis process are shown in Table I.

The average surface roughness (Ra) of the DLC-coatedspecimens was determined using a TalySurf 6 unit (RankTaylor Hobson Ltd., Leicester, UK). The mean (6 SD) forn 5 4 specimens of DLC-coated polystyrene and DLC-coatedcobalt-chromium were 0.036 (6 0.003) mm and 0.015 (60.006) mm, respectively, as compared with mean values of0.036 and 0.039mm for uncoated polystyrene and cobalt-chromium, respectively.

Uncoated and DLC-coated polystyrene and metal sampleswere sterilized in ethylene oxide prior to use. A period of atleast 15 days was allowed between sterilization and implan-tation to allow for complete diffusion of ethylene oxide fromthe test samples.

Experimental Design: In Vitro Studies

Cell suspensions were prepared in either McCoys 5A medium(for SaOS-2) or DMEM (for MG-63). For experimental work,basal media were supplemented with 2% fetal calf serum(FCS), 50 IU/mL penicillin, 50mg/mL streptomycin, 2mML-glutamine, 10 nM vitamin K3, and 50mg/mL ascorbic acid.Ascorbic acid and vitamin K3 are required for the expressionof type I collagen and osteocalcin, respectively.26

Cells were seeded into 24-well plates at density of 53 104

cells per well. After 24 h in the incubator, the medium wasremoved and the cell monolayer washed with sterile phos-

TABLE I. Deposition Parameters for Fast Atom Bombardment and Plasma-Assisted Chemical Vapor Deposition Processes Used toPrepare DLC Coatings

OperatingTemperature (°C) Power Output (W) Precursor Gas Deposition Time (h) Film Thickness (mm)

Fast Atom Bombardment 40–60 400 Acetylene 4 0.50Plasma-Assisted Chemical

Vapor Deposition 125–250 300 Hexane 1 1.80

320 ALLEN, MYER, AND RUSHTON

phate buffered saline (PBS). Fresh medium, this time supple-mented with 10 nM 1a, 25-dihydroxyvitamin D3, was thenadded and the plate returned to the incubator.

Samples of culture medium were harvested 24, 48, and72 h after the addition of vitamin D3. Aliquots were dis-pensed into Eppendorf tubes for storage at either14 °C (forlactate dehydrogenase assays) or270 °C (for osteocalcin andtype I collagen assays). Cell lysates were prepared by theaddition of 1% (v/v) Triton X-100t (BDH, Poole, UK) inPBS to the cell monolayer and were stored at14 °C forlactate dehydrogenase and alkaline phosphatase assays.

Cytotoxicity Assays

A commercial assay kit (CytoTox96; Promega Corporation,Madison, WI) was used in this study. For each test sample,the percentage LDH release was calculated by dividing theamount of LDH in the medium sample by the total amount ofLDH in the medium and cell lysate. LDH release has beenshown to provide a sensitive and accurate index of celldeath.27

Quantification of Cell Numbers

LDH activity in cell lysates was used to determine the cellconcentration in the lysate.28 Plots of cell number vs. timewere constructed for both cell lines. The cell number datawere also used to calculate the specific ALP activity.

Cell Morphology

Intact cell monolayers were rinsed in PBS, fixed in methanolfor 10 min, and stained with May Grunwald Giemsa. Cellswere then examined by transmitted light microscopy.

Alkaline Phosphatase Assay

Alkaline phosphatase (ALP) activity was measured in celllysates using a spectrophotometric technique.29 ALP activi-ties in test samples were corrected for time and cell numberand were expressed as nmolp-nitrophenol per min per mil-lion cells. Because the levels of ALP in MG-63 cells areextremely low,26 only SaOS-2 cells were used for ALP as-says.

Synthesis of Type I Collagen

The synthesis of type I collagen was quantified with a com-mercial radioimmunoassay for the carboxyterminal propep-tide of the procollagen type I molecule (Procollagen PICP;Orion Diagnostica, Espoo, Finland). This is a competitiveassay in which PICP in the test sample competes with125I-labeled PICP for binding sites on a rabbit anti-PICP antibody.The amount of free (unbound) radiolabeled PICP is inverselyproportional to the concentration of PICP in the test sample.The concentration of PICP was determined from a standardcurve. The assay has a sensitivity of 1.2mg/L and can detectPICP concentrations in the range 25–500mg/L.

Osteocalcin Release

The concentration of osteocalcin in culture supernatants wasdetermined with a commercial enzyme-linked immunoassay(Osteocalcin ELISA Kit; DAKO Ltd., High Wycombe, UK).This is a competitive assay in which osteocalcin in the testsample and biotinylated osteocalcin in the assay reagentcompete for binding on wells coated with anti-osteocalcinantibody. Peroxidase-conjugated streptavidin is then addedand binds to the biotinylated osteocalcin. The streptavidinconverts a colorless chromogenic substrate into a coloredproduct whose absorbance can be determined spectrophoto-metrically. The concentration of osteocalcin in test sampleswas calculated from a standard curve. Because the levels ofosteocalcin synthesis in SaOS-2 cells are much lower than inMG-63 cells, only the latter were tested.

Surgical Procedures

Four male Sprague–Dawley rats (500–600 g) were used inthe intramuscular implantations. The rats were anesthetizedwith an intraperitoneal injection of a 1:1:2 mixture of fenta-nyl-fluanisone and midazolam in sterile water, and the leftand right hind limbs clipped and prepared for aseptic surgery.Stab incisions were made in the quadriceps muscle with a #15blade, and fine scissors were then used to create a smallpocket within the muscle. The uncoated or DLC-coated spec-imen was gently inserted into the intramuscular pocket,which was then repaired with a single resorbable suture. Theskin incision was closed with three resorbable sutures. Theprocedure was then repeated for the contralateral limb. Theuncoated cobalt-chromium alloy cylinder was always im-planted into the left quadriceps, the DLC-coated specimen inthe right quadriceps. Animals received a single intramuscularinjection of clavulanate-potentiated amoxicillin at the time ofsurgery. No attempt was made to limit their activity aftersurgery.

Figure 1. Transcortical implants in the lateral femoral cortex ofsheep. Two (black) DLC-coated implants and one (silver) uncoatedcobalt-chromium alloy implant are shown in this intraoperative pho-tograph.

321BIOCOMPATIBILITY OF DLC

Two adult female Mule ewes (70–85 kg body weight)were used in the intraosseous studies. Animals were fastedfor 24 h and then premedicated with an intramuscular injec-tion of xylazine (0.22 mg/kg). General anesthesia was in-duced with an intravenous injection of ketamine (11 mg/kg)and maintained with an inhaled mixture of halothane (1.5–3%) in oxygen. Left and right hind limbs were clipped,scrubbed, and draped for aseptic surgery. A straight skinincision was made over the lateral femur, and subcutaneoustissues and muscle were incised and retracted to expose theperiosteal surface of the femur. Five drill holes were preparedwith a 4.0-mm stainless steel drill bit; each drill hole ex-tended through the lateral cortex of the femur. The implants(3 DLC-coated and two uncoated specimens) were insertedinto the drill holes in press-fit manner. Care was taken toensure that the end of the implant was level with the outercortex of the bone (Fig. 1). The surrounding muscles wererepositioned, subcutaneous tissues repaired with resorbable

sutures, and the skin incision closed with stainless steelwound staples. The procedure was then repeated for thecontralateral limb. Ampicillin (25 mg/kg IV) was adminis-tered at the time of induction, and flunixin meglumine(1.1mg/kg IV) was given to control postoperative discomfort.

Histopathology

Animals were euthanized 12 weeks after surgery. Left andright quadriceps muscles (rats) and femora (sheep) wereharvesteden bloc and fixed in buffered formalin. Sampleswere then sequentially dehydrated in ethanol, embedded inepoxy resin, and sectioned to 350mm with a low-speed sawequipped with an aluminum oxide blade. Transverse sectionsof the implant-tissue interface were ground to a thickness ofapproximately 175mm, surface polished, etched, and stainedwith 0.25% toluidine blue (pH 9.0). Specimens were exam-ined under a light microscope and the thickness of the fibrousinterface measured at three sites chosen at random.

Figure 2. Morphology of osteoblastic cells. SaOS-2 cells on (A) polystyrene and (B) DLC-coatedpolystyrene; MG-63 cells on (C) polystyrene and (D) DLC-coated polystyrene. May Grunwald Giemsastain; original magnification: 3 100.

322 ALLEN, MYER, AND RUSHTON

Statistical Analysis

In the cell culture studies, triplicate samples were tested ineach experiment. Each experiment was performed twice. Theunpaired Student’st-test was used to determine statisticalsignificance atp , 0.05. Histopathology results were com-pared with the paired Student’st-test. A significance level ofp , 0.05 was considered statistically significant.

RESULTS

Cell Morphology

Light microscopy confirmed that DLC coatings had no ad-verse effect on the morphology of osteoblast-like cells (Fig.2). There was no evidence of cell death, abnormal cell stain-ing, or cytoplasmic vacuolation.

Cytotoxicity

There were no statistically significant differences in cytotox-icity for cells growing on the uncoated and DLC-coatedplates (Fig. 3).

Cell Growth

When equivalent numbers of SaOS-2 and MG-63 cells (53104 cells) were seeded onto polystyrene and DLC-coatedpolystyrene plates, initial cell growth appeared to be betterfor the MG-63 cell line, as indicated by the greater cellnumber at 24 h (Fig. 4). However, once a stable population ofcells had become established, growth rates were similar for thetwo cell lines. For both SaOS-2 and MG-63 cells, growth rateson the uncoated and DLC-coated substrates were equivalent.

ALP Activity

MG-63 cells did not produce detectable levels of ALP. ALPactivity in SaOS-2 cells was stimulated by 1a, 25-dihy-droxyvitamin D3, and cells growing on both the uncoated andDLC-coated polystyrene produced high levels of ALP activ-ity. There were no significant differences between uncoatedand coated substrates (Fig. 5).

Osteocalcin

Osteocalcin was detected in medium from MG-63 cells butnot SaOS-2 cells. As expected, the concentration of osteocal-cin in the culture medium increased over the 72-h incubationperiod, but there were no significant differences between cellson the two substrates (Fig. 6).

Synthesis of Type I Collagen

SaOS-2 and MG-63 cells produced detectable quantities ofPICP in culture. PICP concentrations were higher in MG-63cells than in SaOS-2 cells, but there were no significantdifferences between the results for uncoated and DLC-coatedpolystyrene for either cell line (Fig. 7).

Histopathology

Uncoated and DLC-coated cobalt-chrome implants were welltolerated in muscle. There was no evidence of discolorationof tissues around the implant. A thin layer of fibrous tissueseparated the implant from the surrounding muscle (Fig. 8).There was no statistically significant difference between thethickness of the fibrous layer around plain and DLC-coatedimplants (Table II).

Although attempts were made to quantify the osseousresponse to the DLC and CoCr implants, this proved to beextremely difficult, because the majority of implants hadmigrated into the cortical bone and were no longer flush withthe bone surface. Qualitative analysis of sections through thebone–implant interface revealed evidence of reactive boneformation within all three bone envelopes (periosteal, intra-cortical, and endosteal).

In the periosteal envelope, a marked periosteal reactionhad developed in all cases, with lifting of the periosteum andformation of new (primarily woven) bone on the outer surfaceof the cortex. This new woven bone filled and obscured theoriginal drill holes in the cortex.

Migration of the implant towards the inner surface of thecortex had reduced the effective area of contact between theimplant and adjacent bone within theintracortical envelope.

Figure 3. Cytotoxicity results for (A) SaOS-2 and (B) MG-63 cellsgrowing on uncoated and DLC-coated polystyrene.

323BIOCOMPATIBILITY OF DLC

However, the defect in the bone tunnel had been repaired bynew bone formation and, as a result, there was a significantamount of bone–implant contact. For both the DLC-coatedand uncoated CoCr, the interface between the implant andthis new cortical bone appeared to be mature, with bone andmetal separated by only a thin layer of fibrous tissue (TableIII and Fig. 9). Small collections of yellow-gold particulatedebris were seen in 3 of 34 sections through the bone–implantinterface. This debris was golden yellow in color and wasassociated with a mild fibrous reaction.

At the endostealsurface, a collar of new bone stabilizedthe implants. The interface between the newly formed en-dosteal bone and the surface of the implant appeared matureand healthy, with only a very thin layer of fibrous tissue andno evidence of inflammation.

DISCUSSION

There have been several reports on investigations into thebiocompatibility of DLC coatings in recent years.30–35Many

of these studies were performed by research groups interestedin cardiovascular applications for DLC, and for this reasontheir investigations focused on issues of cytotoxicity andthrombogenicity. The consensus from all this testing has beenthat DLC coatings are noncytotoxic and nonthrombogenic.However, specific information about the effects of DLC coat-ings on cellular metabolism has been relatively scarce, andeven less information is available as to the behavior of DLCcoatingsin vivo.

Because our interest has been in the potential use of DLCas a wear-retardant coating for total joint replacements, it waslogical for us to focus attention on a systematic analysis of theinteractions between DLC and the cells/tissues of the mus-culoskeletal system. Preliminary cytotoxicity testing of DLCin vitro indicated that the material is well tolerated by mac-rophages, fibroblasts, and osteoblasts,33,34 three cell typesthat are typically found within periprosthetic tissues. Metallicand nonmetallic substrates coated with DLC have also beenshown to support the adhesion and growth of osteoblasticcells in vitro.36–38However, none of these studies has exam-

Figure 4. Plots of cell number over time for (A) SaOS-2 and (B)MG-63 cells growing on uncoated and DLC-coated polystyrene. At24 h, more SaOS-2 cells are present compared with MG-63 cells,suggesting enhanced attachment and/or proliferation, but the MG-63cells subsequently proliferated at a faster rate. For both cell lines,growth on the DLC-coated substrate was similar to that on theuncoated substrate.

Figure 5. Alkaline phosphatase activity in SaOS-2 cells growing onuncoated and DLC-coated polystyrene.

Figure 6. Osteocalcin production by MG-63 cells growing on un-coated and DLC-coated polystyrene.

324 ALLEN, MYER, AND RUSHTON

ined in detail the effects of DLC coatings on the syntheticactivity of osteoblasts. The goal of the present study was,therefore, to extend the study of osteoblastic growth on DLCto include a systematic analysis of both cellular growth andmetabolism. For this purpose, we elected to focus on anosteoblastic culture system in which osteoblast-specific pro-tein synthesis can be quantified and compared under differentculture conditions. The model that we used has been charac-terized extensively in our laboratory.26,29,34,39,40Moreover,osteoblast cultures are widely accepted as a standardin vitromodel for investigating the biological effects of orthopedicbiomaterials, and a large number of studies have been pub-lished in which osteoblast cultures have been used to estab-lish the biocompatibility41–48or possible toxicity39,49of can-didate materials

In the present study, the two cell lines (MG-63 andSaOS-2) attached to and proliferated on both the uncoatedand DLC-coated polystyrene substrates. Analysis of culturemedium and cell lysates showed that the cells maintained anosteoblastic phenotype on the DLC coating, with expressionof alkaline phosphatase, osteocalcin, and type I collagen.Expression of each of the three markers was similar onuncoated and DLC-coated polystyrene; because the tissueculture grade polystyrene that was used in these experimentsis known to be biocompatible, one can conclude that the DLCcoating is also biocompatiblein vitro.

The histopathological analysis of implants retrieved fromintramuscular and intraosseous sites revealed that the DLC-

TABLE II. The Effects of Implant Type on the Thickness of theFibrous Reaction Around Intramuscular Implants

ImplantType

ExperimentalPeriod(weeks)

Number ofSections

Thickness ofFibrous

Layer mm(mean6 SD)

Uncoated 12 8 53.56 22.0DLC-coated 12 8 48.06 17.4

TABLE III. Fibrous Reaction to Intraosseous Implants

ImplantType

ExperimentalPeriod(weeks)

Number ofSections

Examined

Thickness ofFibrous

Layer mm(mean6 SD)

Uncoated 12 18 27.46 15.5DLC-coated 12 16 29.66 9.3

Figure 7. Release of PICP from (A) SaOS-2 and (B) MG-63 cellsgrowing on uncoated and DLC-coated polystyrene. Only 72-h datawere collected for SaOS-2 cells.

Figure 8. Representative sections through the implant–muscle inter-face in (A) uncoated and (B) DLC-coated specimens. Toluidine bluestain; original magnification: 3 20.

325BIOCOMPATIBILITY OF DLC

coated implants were well tolerated. In both sites, implantswere surrounded by a thin fibrous membrane, with no evi-dence of acute inflammatory reactions or cellular necrosis.These histological findings are typical of an inert biomaterial.The reactions to the uncoated and DLC-coated implantwere indistinguishable and, because CoCr is recognized as

having excellent biocompatibility, one must infer that theDLC-coated implant also exhibits excellent biocompatibil-ity. Recent work by Guglielmotti et al. using a short-term(30-day) implantation protocol in rat bone has also dem-onstrated the biocompatibility of DLC-coated ceramic de-vices in vivo.50

Although the intraosseous implants migrated into the cor-tex after implantation, all had become stabilized by a collar ofnew bone on the endosteal surface of the lateral cortex.Similar results have been reported by Luckey et al., whofound that osseous attachment to smooth cobalt-chromiumimplants was poor in both cancellous and cortical sites.51 Asin our study, histological analysis confirmed that direct con-tact with bone was limited to regions where callus had de-veloped in response to the implant.

Particulate debris was identified in 3 of the 34 sections (2from the uncoated CoCr group, 1 from the DLC group). Thisdebris was extracellular, with an appearance that was notconsistent with hemosiderin. It is most likely that the debrisrepresents a corrosion product, either from the implant or,more likely, from the stainless steel drill bit used to create thehole in the cortex. The debris was surrounded by a mildfibrous reaction, with little evidence of cellular activity. Sim-ilar changes have been reported in human tissues retrievedfrom around stainless steel implants.52 Unfortunately, wewere not able to perform elemental analysis on the threesections, so the exact nature of this material remains un-proven.

Taken together, the results from thesein vitro and in vivostudies confirm the excellent biocompatibility of DLC-coatedsubstrates. Based on these results, we have initiated a long-term animal study to investigate the tribological and biolog-ical performance of a DLC-coated knee arthroplasty implantin sheep.

This work was supported by Howmedica International, Staines,UK.

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